BioHelix is the exclusive licensee for a novel, primase-based Whole Genome Amplification (pWGA) technology invented by Drs. Stanley Tabor and Charles Richardson at Harvard Medical School. This pWGA system utilizes multiple replication proteins including a primase/helicase, a DNA polymerase, a single-stranded DNA binding protein, and several other accessory proteins to achieve rapid and sensitive whole genome amplification. As such, bringing this novel technology from the university laboratory to the market place is a challenging task. During Phase I, we evaluated the performance of this technology and successfully purified all necessary proteins in large production scale. We successfully launched the first generation Rapisome" pWGA kit, which is, to our knowledge, the first commercially available DNA amplification system reconstructed from a cellular replisome. We have also discovered additional advantageous features of the pWGA platform. First, we have shown that the pWGA platform can perform real-time detection of trace amounts of DNA (100 fg). In addition, we have demonstrated that in comparison with another commercially available WGA product (GenomiPhi from GE Healthcare) the pWGA platform is particularly efficient in amplifying (by 108 fold) circular DNA from low copy numbers (100 copies) of input. The overall goal of the Phase II research will be: 1) To continue to improve the performance of the pWGA system by optimizing the key components and fine-tuning each component (if successful, a second-generation pWGA kit with lower amplification bias and higher fidelity will be launched); 2) To explore new applications for pWGA in biomedical research and clinical diagnostics based on the aforementioned studies in Phase I. Based on the phase I data, the applications of pWGA may be expanded into two new areas. One is as a universal DNA detection system, which will be developed for the detection of trace amounts of DNA contamination in biological samples or pharmaceutical products. The second new application is related to circular DNA amplification, based upon the phase I observation that pWGA can amplify circular DNA with extremely high efficiency (from 100 copies to over 1010). We propose to develop pWGA as a tool for clinical research and diagnostic applications targeting circular DNA viruses such as human Papillomavirus (HPV). We will use pWGA to amplify samples containing HPV and subsequently determine the HPV genotype using the Luminex system. PUBLIC HEALTH RELEVANCE: Whole Genome Amplification (WGA) technologies are useful tools for cancer and genetic research. Amplifying the entire genome enables researchers to perform more tests on a given sample than would otherwise be possible. Two other types of WGA have been commercialized for research applications: 1) methods derived from the polymerase chain reaction and 2) multiple displacement amplification (MDA). Rubicon Genomics has developed a PCR-based WGA platform GenomePlex" Kits for Research Use, available through Sigma-Aldrich. GE Healthcare markets the MDA technology under the name GenomiPhi" while Qiagen sells MDA kits under the name REPLI-g(R). Both of these technologies have some limitations. The GenomePlex" system generates short PCR amplicons, which limits its use in downstream applications such as RELP, DNA sequencing, and cloning. In addition, it requires a multiple-step reaction setup. MDA also often requires a heat-denaturation step before isothermal DNA amplification to facilitate primer annealing, which increases the complexity of the reaction setup and may introduce mutations into the template. In addition, the use of random primers in MDA makes it prone to non-specific DNA amplification even in the absence of an input template. The primase-base Whole Genome Amplification (pWGA) platform is an in vitro reconstruction of a cellular replisome. It performs a simple, one-step isothermal whole genome amplification without added primers. The pWGA reaction is more rapid than either PCR or MDA. The amplification is highly sensitive and specific for input DNA template. Amplification of a minimal amount (100 fg) of input DNA can be distinguished from background, which has a potential of being developed into a fast universal DNA detection tool for detection and quantification of unwanted DNA contamination in a given sample. In comparison with MDA, pWGA is 100-times more efficient in amplifying circular DNA, especially when the amount of input is limited (100 copies). We believe that this feature of pWGA can be used to develop a highly sensitive multiplex assay for the detection and genotyping of a broad spectrum of circular DNA viruses, such as human Papillomavirus (HPV) and Herpes Simplex Virus (HSV). [unreadable] [unreadable] [unreadable]